it should be possible to monitor both the induction of the hemolysin in the bacteria as itinfects the host, and the host’s response to this bacterium with regard to its IL-6 induction.In addition to a large number of infectious disease (9, 10, 14) and oncology (4, 7, 25) ani-mal models that have been developed at Xenogen, an extensive program has also beenestablished for the generation of transgenic animals expressing firefly luciferase, designatedas LPTA
animal models, under the control of different inducible promoters [e.g., induciblenitric oxide synthase promoter, VEGFR2 promoter, rat insulin promoter, heme oxygenasepromoter (6, 37) and bone morphogenesis protein 4 promoter (36)]. These LPTA
animalmodels allow the effects of a particular compound (chemical or biological) to be visualizedin the whole animal as that compound is absorbed and metabolized by the differenttissues/organs of that animal. Thus, multiple data points can be collected over time andfrom different regions (tissues/organs) within the same animal.
Background on RNAi Research
Since the first successful report of the use of small interfering RNA (siRNA) to silence geneexpression in mammalian cells (8), a flood of papers reporting the use of RNA interference(RNAi) to elucidate mammalian gene function has followed. Delivery of synthetic siRNAsto mammalian cells in culture can be achieved using lipophilic agents or electroporation.Alternatively, interfering RNAs can be expressed from a plasmid harbored by the cells ofinterest, in which pairs of short complimentary RNA molecules (17, 23, 35), or a singleinverted small hairpin RNA (shRNA) are stably expressed and used for RNAi gene silencing(1, 22, 30, 35). However, the efficiency of transfection depends on the cell type, as doesthe ability of a given siRNA to silence a particular gene, making interpretation of RNAiexperiments difficult.The use of RNAi in living mice has also been widely reported (2, 12, 18, 20, 21, 28, 29, 31,32, 34), fueling hope that siRNAs may one day be used to treat human diseases. Again, twostrategies for the introduction of RNAi molecules have been used for animal experiments:synthetic siRNA or shRNA delivered directly, or delivery of a plasmid or viral siRNA/shRNAexpression cassette that potentially provides a more stable and long lasting delivery of theRNAi species. Although luciferase reporters were used in a number of these studies (18, 20,21, 32), green fluorescent protein has also proven popular as an alternative reporter (2, 12,18, 28, 31, 34). However, whereas the use of luciferase has allowed quantitative non-invasiveanalysis of gene suppression in live animals, studies using GFP as a reporter have requiredex vivo tissue extraction or cell rescue and FACS to allow visualization of GFP suppression.Moreover, quantification can only be accurately achieved using Northern analysis, whichare time consuming and require sacrifice of the experimental animals. Similarly, detectionof RNAi effects on specific host gene suppression (12, 28, 29) have again required FACS,Northern and western analysis of host tissue and cells.Xenogen’s biophotonic imaging technology provides an ideal strategy to non-invasivelymonitor RNAi in small mammals. In 2002, McCaffrey et al. at Stanford University (20, 21)reported the success of both siRNA and shRNA approaches to reduce luciferase expressionin mice following hydrodynamic transfection methods to introduce the RNAi and luciferase
Xenogen In Vivo RNAi Applications
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